Development of the first European Xanthomonas euvesicatoria pv. euvesicatoria lytic bacteriophage cocktail effective in controlling bacterial spot disease in pepper plants.

Bacterial spot of pepper and tomato is one of the most challenging xanthomonad diseases, affecting widely cultivated horticultural crops. Xanthomonas euvesicatoria pv. euvesicatoria (Xee), the main causal agent of bacterial spot in pepper, causes significant yield losses worldwide and is increasingly being reported in Southern Europe, including Spain, where outbreaks of bacterial spot have mainly been detected in pepper. Current management strategies mainly rely on copper-based compounds with limited efficacy due to the development of resistance and environmental and health concerns. Bacteriophages represent a promising safe and sustainable ecological alternative. In this study, lytic phages infecting Xee were isolated from pepper plots affected by bacterial spot disease in two distant regions of Spain. Genomic and taxonomic analysis of five selected phages classified them as belonging to the genus Beograduvirus and confirmed their lytic nature and safety. Three phages were investigated further because of their ability to infect multiple strains of the pathogen while remaining highly specific. These phages exhibited myovirus morphology and remained stable across temperatures and pH conditions relevant to field application. In vitro co-culture of the target bacteria with the three phages, either individually or in combination, revealed that the three combined phages controlled pathogen growth significantly better than single-phage treatments. In pepper plants, foliar application of the phage cocktail by dipping or spraying markedly reduced disease symptoms after inoculation with Xee, with spraying giving the most consistent results regardless of the bacterial strain tested. This is the first European phage cocktail that can effectively control Xee in pepper plants, highlighting its potential as a practical tool for the integrated management of this disease.